The locations of recombination hotspots in humans and mice and their frequencies of undergoing genetic crossing over determine patterns of linkage disequilibrium and the possibilities for closely linked genes to be co-inherited;both are critical issues in efforts to identify genes important in human health and disease. Our experiments have revealed the existence of a hitherto unknown, macromolecular regulatory system that controls the location and relative activity of mammalian recombination hotspots by activating, suppressing and modulating their activity. Genetic variation in this regulatory system first enabled its discovery and now provides a means of identifying its components and their interactions, an essential step in understanding its mechanisms, as well as its relevance to issues of human genetics, population biology and evolution. The first regulatory protein we identified in this way, PRDM9, enables recombination at a family of mouse hotspots by modifying chromatin structure, allowing formation of the initiating double strand break. Others have simultaneously identified PRDM9 as a regulator of human recombination hotspots. In this project we will identify components of the complementary regulatory system controlling suppression of recombination at specific hotspots, a matter of equal concern in understanding the regulation of recombination. We will identify the genes encoding suppressors of five hotspots on mouse Chr 1 whose activities are regulated by genes that differ between the M. m. domesticus strain C57BL/6J (B6) and the M. m. castaneus strain CAST/EiJ (CAST) by: (a) applying a new, considerably improved, quantitative assay system that measures hotspot activities in sperm DNA samples using NextGen DNA sequencing;(b) using this assay to map and clone the regulatory genes involved;(c) testing their interactions with each other and whether they act in a dose dependent manner, indicating whether they act catalytically or stoichimetrically;and finally, (d) testing whether they control the initiation of recombination or the decision between the alternative recombination pathways leading to crossing over v. non-crossover gene conversions. In separate experiments we will also lay the groundwork for identifying additional regulatory factors with allelic differences between M. m. domesticus (B6) and the M. m. musculus strain PWD. We expect to learn whether each hotspot has its own unique regulatory system or whether there are shared regulatory elements, what these molecules are, whether controls are exerted on the initiation of recombination or the choice between alternate pathways of recombination, and the manner in which any of these genes interact with each other. These data together with the molecular identity of these genes will provide information essential to resolving their mechanism of action. The results will considerably enhance our understanding of one of the most basic of biological processes, genetic recombination. PUBLIC HEALTH RELEVANCE: Proper genetic recombination is essential for successful reproduction in all sexually reproducing organisms, including humans. It assures the orderly segregation of chromosomes at meiosis is an important feature of evolutionary processes and generates the genetic diversity that makes each of us a unique individual. Any failure of the recombination process results in sterility. An important feature of human and mouse recombination is the location of genetic crossovers at specialized sites along chromosomes called hotspots. We now understand that there is a macromolecular regulatory system controlling the location and activity of hotspots, and it is this system that we will be studying in this project with the intent of understanding its role in allowing proper recombination.